The goal of this study is to understand at a molecular level how cells make decisions on what action to take when faced with environmental stress. This is a universally important question, as many types all cells have to decide how to handle adverse environmental conditions on a consistent basis. To study this we use the model system S. cerevisiae. In this system, following oxidative stress, yeast (like all eukaryots) has to make a decision. They can stop dividing, assess the situation, repair any damage and then re-initiate cell division. Alternatively they can initiate cell suicide via programmed cell death (PCD). In thi proposal how the cell translates unfavorable environmental conditions into a molecular decision will be studied. Cyclin C is a non-cycling cyclin that is part of the mediator complex that plays a important role in this decision process. Cyclin C is a transcription factor which along with its kinase partner Cdk8, represses a subset of stress responsive genes. To relieve this repression, cyclin C, but not Cdk8p, translocates from the nucleus to the cytoplasm in cells exposed to pro-oxidants and other stressors. In the cytoplasm, cyclin C interacts with the fission machinery and is both necessary and sufficient to induce extensive mitochondrial fragmentation. In addition, cyclin C has a cytoplasmic role in promoting PCD. These results suggest that cyclin C function connects mitochondrial fission to the cell death pathway. This it achieves by inducing stress-induced mitochondrial hyper-fission, which is the first step in the PCD pathway. Therefore, whether or not to release cyclin C into the cytoplasm represents a key life or death decision for the cell. My lab has recently identified that the cell wall integrity MAPK pathway transmits this extra-cellular stress signal directly to cyclin C. This work has shown that the molecular switch that mediates cyclin C release is more complicated than simply an on-off phosphorylation event. Preliminary data has also revealed that Med13p, a member of the mediator complex, is involved in this decision, as this protein is required to retain cyclin C in the nucleus in un-stressed cells. Lastly, we have also identified that a second signaling AMP pathway is also required for cyclin C release. Unlike the CWI pathway this pathway does not directly phosphorylate cyclin C, but rather activates Ask10p, an event that is required for cyclin C release. Thus the focus of this proposal is analyze, at a molecular level, how this second pathway transmits to stress signal to cyclin C as well as to decipher the role Med13p plays in this important molecular switch. Two aims are proposed. Aim 1. Define the mechanism(s) dissolving Med13p interaction allowing nuclear release of cyclin C. This aim will utilize genetic and biochemical approaches to determine what role Med13p destruction and cyclin C phosphorylation play in mediating translocation of the cyclin. Aim 2. Dissect the AMP pathway stimulating cyclin C translocation. This aim will employ genetic and cell biological approaches to dissect how this pathway stimulates cyclin C relocalization.